Plague is a zoonosis that is present in wild rodent populations worldwide and is transmitted primarily by fleas. Yersinia pestis, the plague bacillus, is unique among the enteric group of gram-negative bacteria in having adopted an arthropod-borne route of transmission. Y. pestis has evolved in such a way as to be transmitted during the brief encounter between a feeding flea and a host. A transmissible infection depends on the ability of Y. pestis to grow in the flea as a biofilm that is embedded in a complex extracellular matrix. Bacteria in the biofilm phenotype are deposited into the dermis together with flea saliva, elements which cannot be satisfactorily mimicked by needle-injection of Y. pestis from laboratory cultures. The objective of this project is to identify and determine the function of Y. pestis genes that mediate flea-borne transmission and the initial encounter with the host innate immune system at the infection site in the skin. We study the interaction of Y. pestis with its insect vector by using an artificial feeding apparatus to infect fleas with uniform doses of wild type or specific Y. pestis mutants. We seek to identify Y. pestis genes that are required for the bacteria to infect the flea midgut and to produce a biofilm that blocks the flea foregut and that is required for efficient transmission. The strategy entails first identifying bacterial genes that are differentially expressed in the flea by gene expression analysis and other techniques. Specific mutations are then introduced into these genes, and the mutants tested for their ability to infect and block the flea vector. Identification of such transmission factors allows further studies into the molecular mechanisms of the bacterial infection of the flea vector. Detailed understanding of the interaction with the insect host may lead to novel strategies to interrupt the transmission cycle. During the last year, we identified and characterized all the Y. pestis genes predicted to encode enzymes that either synthesize or degrade the bacterial second messenger c-di-GMP, a key regulator of the biofilm life stage of Y. pestis in its flea vector. We proved that Y. pestis has only two functional genes that function to synthesize c-di-GMP. We went on to show that the relative contribution of the two genes to the biofilm phenotype is influenced strongly by environmental niche one is most important during infection of the flea, and the other predominates during in vitro growth. We also continue to investigate the genetic changes that led to the evolutionarily recent transition of Y. pestis to an arthropod-borne transmission route. Many nonfunctional genes (pseudogenes) occur on the Y. pestis genome;some of them are implicated in regulating the biofilm phenotype that enhances transmission by fleas. We have found that restoration of three pseudogenes with the equivalent functional genes of Yersinia pseudotuberculosis, the recent ancestor of Y. pestis, reduces transmissibility in the flea;and conversely, mutation of these genes in Y. pseudotuberculosis results in the gain of biofilm-forming ability in the flea. Thus, gene loss appears to have played a significant role in Y. pestis evolution. We have also further developed models to examine host-parasite interactions in the dermis after transmission by flea bite, and are completing a project on the host immune response to flea saliva and how this influences transmission. We have established a colony of the ground- and rock-squirrel flea Oropsylla montana, an important vector of plague in the U.S. We intend to compare transmission efficiency and the biofilm-dependent blockage ability of Y. pestis in X. cheopis and O. montana.